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1.
Transgenic plants of Jincheng orange (Citrus sinensis Osbeck) and Newhall navel orange (C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange (Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease.  相似文献   

2.
Sweet orange (Citrus sinensis L. Osb.) ‘Hamlin’ is a canker (Xanthomonas axonopodis pv. citri: Xac) susceptible citrus genotype grown commercially worldwide. Canker causes severe economic losses and restricts the marketability of crop for export. Little is known about the role of oxidative stress in canker development. In the present investigation, sweet orange ‘Hamlin’ leaves were artificially inoculated with Xac to determine the impact of Xac infection on hydrogen peroxide (H2O2) metabolism. Characteristic symptoms following artificial inoculation were water soaking of the infiltrated zone between 2 and 8 days after inoculation (dai); raised epidermis accompanying tiny yellow colored bacterial colonies at 8 dai; and yellowing and necrosis of the infected zone by 12–16 dai. In planta Xac population increased 1000 fold by 14 dai from an initial population of 7.3 × 106 cfu cm−2 (0 dai). Peak concentrations of H2O2 were observed at 24 h and between 8 and 10 dai and coincided with higher activity of total superoxide dismutase (SOD). Lower levels of H2O2 in infected leaves were maintained by Xac induced higher activities of catalase (CAT), ascorbate peroxidase (APOD), and guaiacol peroxidase (POD). It appears Xac altered H2O2 metabolism in C. sinensis L. Osb. ‘Hamlin’ to enhance survival and growth.  相似文献   

3.
Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin (Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin (Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin (C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 106 CFU mL−1X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime (Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 1010 CFU mL−1) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.  相似文献   

4.
Asiatic citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most detrimental diseases affecting citrus production. ‘Meiwa’ kumquat (Fortunella crassifolia) and ‘Newhall’ navel orange (Citrus sinensis) are two genotypes with contrasting Xcc resistance. In the current study, efforts were made to perform pairwise comparison between them in terms of structural difference and biochemical response to Xcc attack. Pinprick inoculation demonstrated that ‘Meiwa’ had lower disease incidence rate and smaller lesion size, concurrent with significantly less bacterial number at the inoculation sites relative to ‘Newhall’. Microscopic observation demonstrated that stomatal density and size of ‘Meiwa’ were significantly smaller than ‘Newhall’. Stomatal apertures of ‘Meiwa’ were smaller than ‘Newhall’ measured at 2 h after their leaves were immersed in Xcc suspension, consistent with the bacteria number in the internal tissues counted at 4 d. Epicuticular wax content of ‘Meiwa’ was significantly higher than that of ‘Newhall’, while no difference in palisade and spongy tissue was observed between them. Enzymatic activity assay showed that after Xcc inoculation, ‘Meiwa’ exhibited significantly higher activities of catalase (CAT), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in comparison with ‘Newhall’ at most of the sampling time points. Our data showed that the two genotypes with contrasting Xcc resistance exhibited clear difference in the structure and physiological response, which may provide information to facilitate our understanding of Xcc resistance.  相似文献   

5.
In order to reduce unnecessary amount of P-fertilizer and severity of Phytophthora root rot in citrus orchards, the experiment was set up. Thirteen indigenous arbuscular mycorrhiza (AM) fungi species were isolated from rhizosphere soil of citrus orchards in Thailand and were then propagated into three host plants [sorghum (Sorghum bicolor), maize (Zea mays), and leek (Allium cepa)] by trap culture. We also tested whether indigenous AMF species (13 different species) could colonize into three cultivars of citrus scions and rootstocks (Shogun: Citrus reticulata Blanco cv. Shogun; Tangerine: C. reticulata; and C-35 citrange: Citrus sinensis × Poncirus trifoliata). With root colonization rates, the results indicated that Acaulospora tuberculata and Glomus etunicatum provided the best colonization in all citrus cultivars. We selected, therefore, those AMF species to verify their influences on citrus growth and Phytophthora root rot resistance. Three cultivars of citrus scions and rootstocks, Shogun, Tangerine and C-35 citrange, were inoculated with two effective indigenous AMF species, G. etunicatum or A. tuberculata in order to determine the influences on citrus growth. The plants were investigated to determine the mycorrhizal efficiency index (MEI), AM colonization, P content, and other parameters. Co-inoculation of AMF species (G. etunicatum or A. tuberculata) with Phytophthora nicotianae was also carried out in Shogun scion/C-35 citrange rootstocks. The results of citrus growth revealed that Shogun and Tangerine inoculated with G. etunicatum produced the highest MEI. Tangerine and C-35 citrange amended with fertilizers and G. etunicatum showed the highest P content in leaves. This indicated that G. etunicatum has an influence on citrus growth and P uptake, suggesting it to be the highly effective strain. Shogun scion/C-35 citrange rootstock combinations that were inoculated by both P. nicotianae and different AM fungi (G. etunicatum or A. tuberculata) showed root injury at low level of root rot symptom. However, the part of Shogun scions grafted on rootstocks showed severe symptom of shoot die back in treatment inoculated with P. nicotianae alone, while treatment inoculated with different AM species (G. etunicatum or A. tuberculata) and P. nicotianae rendered lower shoot die back symptoms than that of Phytophthora treatment. The low level of shoot die back symptom was shown at first, then healthy young shoot was restored. Our results indicated the facts that different host plants and different AMF species produced different outcomes of growth and pathogen resistance. The application of both AM isolates, therefore, has an enormous potential to be produced the inoculum for citrus orchards.  相似文献   

6.
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7.
Stem nematode (Ditylenchus destructor Thorne) is one of most serious diseases limiting sweetpotato (Ipomoea batatas (L.) Lam.) production, and it is urgent to develop sweetpotato varieties resistant to this disease. In present study, we have developed transgenic sweetpotato (cv. Xushu 18) plants resistant to stem nematodes using oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, uidA gene and hptII gene. Selection culture was conducted using 7 mg/l hygromycin. A total of 2119 plants were produced from the inoculated 1710 cell aggregates of Xushu 18 via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 92.8% of the regenerated plants were transgenic plants. Transgenic plants exhibited enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation and the inoculation test with stem nematodes and stem nematode-resistant plants were selected from the transgenic plants. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 3. High level of OCI gene expression in the resistant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides a way for improving stem nematode resistance of sweetpotato.  相似文献   

8.
Recirculating subirrigation systems are frequently exposed to the risk of plant pathogens transmission, which may deteriorate the growth and quality of the plants. The transmission of Phytophthora nicotianae was examined using Kalanchoe blossfeldiana cv. New Alter in two recirculating subirrigation systems, a nutrient-flow wick culture (NFW) system and an ebb and flow (EBB) system. When the nutrient solution was infested, the pathogen was recovered from roots in both subirrigation systems. However, foliar blights and browning of roots appeared 4 and 7 weeks, respectively, after inoculation in the EBB system. Only a little discoloration appeared in the NFW system. The fresh and dry weights were lower in the EBB system than in the NFW system. When growing medium was inoculated, the pathogen was unable to be isolated from the plants in the NFW system. However, disease symptoms appeared in the EBB system 4 weeks after inoculation, and the pathogen was observed in the basal leaves and roots. Similar to the infested nutrient solution, the plant growth in the EBB system was inhibited. These results suggested that when the nutrient solution was infested, pathogen transmission could occur in plants in both systems, although differences existed with regard to disease symptoms and the time it took for symptoms to appear. However, we observed that when growing medium was inoculated the pathogen was not transmitted to adjacent plants in the NFW system using wick.  相似文献   

9.
Two transgenic strawberry lines (Pel 1 and Pel 3) containing the open reading frame of a fruit specific strawberry pectate lyase gene (FaplC) under the control of the CaMV35S promoter have been obtained to evaluate the role of this gene on fruit softening. Ripen fruits from both lines showed a significant down-regulation of FaplC, being the percentage of silencing of 84 and 71% on Pel 1 and Pel 3, respectively. The agronomic behaviour of transgenic plants was evaluated during two consecutive years. Fruit set increased in the two transgenic lines when compared with control plants, although Pel 1 showed a significant reduction on fruit weight. Firmness of full ripen fruits from Pel lines was significantly higher than control fruits, while color and soluble solids were not affected. The increase of firmness in Pel lines was maintained when ripe fruits were stored for 3 days at 25 °C. Histological analysis of ripe fruits showed lower intercellular spaces and a higher degree of cell to cell contact area in transgenic fruits when compared with controls. Altogether, these results suggest a direct relationship between pectate lyase gene expression and strawberry fruit softening.  相似文献   

10.
Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l−1 6-benzyladenine, 2 mg l−1 zeatin and 1 mg l−1 indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.  相似文献   

11.
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12.
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13.
In the present paper we report on the effects of the insertion of the Agrobacterium rhizogenes rolC gene in the tomato (Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.) cultivar Tondino. Several transgenic lines were successfully obtained, between which two clones, rolC1 and rolC3, were chosen for the analysis of morpho-productive traits as well as of the endogenous levels of auxin and abscisic acid. Consistent with the known phenotypic effect of this gene, the transformed tomato plants were significantly shorter than the corresponding controls. On the other hand, even if yield was not affected by the transformation in terms of average number of fruits produced, fruit weight was significantly lower in the transgenics with respect to the controls. Therefore, insertion of the rolC gene does not lead to an improvement in plant productivity.  相似文献   

14.
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15.
Cucumber (Cucumis sativus L.), which is a vegetable crop, has served as the model system for sex expression in flowering plants, and the inheritance of sex expression in cucumber is well documented. However, the genetics of subgynoecism expression in cucumber had rarely been described. In this study, we investigated the inheritance of subgynoecious traits in cucumber plants with the inbred cucumber lines of subgynoecious (C. sativus L. var sativus cv 97-17 and S-2-98) as the materials. Genetic analysis had showed the two subgynoecious inbred lines were controlled by one pair of recessive gene and one pair of incompletely dominant gene, which were designated presently as mod-F2 and Mod-F1, respectively. Furthermore, the mod-F2 and Mod-F1 loci, which enhance the intensity of femaleness, also inherited independently with F and M genes.  相似文献   

16.
Guilan is one of the main province olive oil producers in Iran. The culture of olive trees is of considerable importance. One problem that affects Iran olive orchards is a disease bacterial canker caused by Pseudomonas syringae pv. syringae. In this research, the bacterium was recovered from sunken brown stem lesions on 2-year-old olive trees cv. Marie, during survey from olive orchards in different areas of Guilan province. Samples were taken from infected tissues and were washed with sterile distilled water and crushed in peptone water. Then 50 μl of the extract was cultured on NA and King's B medium containing cyclohexamid antibiotic (50 μg/ml). After 48–72 h, bacterial colonies were selected. Based on morphological, physiological, biochemical, pathogenicity properties and PCR methods with specific primers the predominant pathogenic type was identified as P. s. pv. syringae. This is the first report of the existence of P. s. pv. syringae on olive trees in Iran.  相似文献   

17.
The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene (hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene (nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5 mg L−1 hygromycin or 75 mg L−1 kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.  相似文献   

18.
Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T0 and T1 plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T0 plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T1 generation based on PCR analysis.  相似文献   

19.
Analysis of polymorphism in ISSR amplicons revealed diverse genetic relationship between Citrus indica and five other citrus species. In a consensus UPGMA dendrogram, based on Nei and Li's distance matrices, the C. indica samples from three different sites of Meghalaya, India were clustered together with 99% bootstrap support. C. reticulata, C. sinensis, C. aurantifolia formed a cluster with 67% bootstrap separation. C. macroptera and C. maxima samples from two sites of Meghalaya formed separate clusters with respectively 100% and 98% bootstrap supports. Principal Components Analysis projected C. indica to be more closely related to C. aurantifolia than to the other citrus species of the study. PCA also exposed variations within the C. maxima and C. macroptera samples.  相似文献   

20.
Agrobacterium-mediated genetic transformation of a tetraploid “tetrazyg” citrus rootstock selection ‘Orange #16’ [Nova mandarin (Citrus reticulata Blanco) + Hirado Buntan pummelo (Citrus grandis L. Osbeck)] × [Cleopatra mandarin (C. reticulata Blanco) + Argentine trifoliate orange (Poncirus trifoliata (L.) Raf.)] was performed. Juvenile epicotyl segments were transformed with a construct containing a bifunctional egfp–nptII fusion gene under the control of an enhanced double CaMV 35S promoter. Our protocol resulted in a reasonable transformation efficiency of 18%. Stable integration of the transgene was confirmed by visual observation of EGFP expression, PCR and Southern blot hybridization. The purpose of this work was to investigate the amenability of novel citrus rootstock germplasm being developed for improved tree size control, soil adaptation, and disease resistance, to existing transformation technologies. Seed trees of such transgenic tetraploids also have potential as trap plants containing potent insecticidal transgenes, due to their inedible fruit and inherent crossing barriers with conventional commercial diploid scion cultivars, and could be planted around producing citrus groves.  相似文献   

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